Analytical cuvette and supply system wherein the cuvette inlet and outlet are located on the bottom of the cuvette



Sept. 17, 1968 E. w. ANTHON 3,401,591

ANALYTICAL CUVETTE AND SUPPLY SYSTEM WHEREIN THE CUVETTE INLET ANDOUTLET ARE LOCATED ON THE BOTTOM OF THE CUVETTE Filed Dec. 9, 1963 2'Sheets-Sheet l INVENTOR. ERIK W. AN THON Sept. 17, 1968 E. w. ANTHON3,401,591

ANALYTICAL CUVETTE AND SUPPLY SYSTEM WHEREIN THE CUVETTE INLET ANDOUTLET ARE LOCATED ON THE BOTTOM OF THE CUVETTE 2 Sheets-Sheet 2 FiledDec. 9, 1965 FIE- 2- United States Patent Ofice Patented Sept. 17, 1968ANALYTICAL CUVETTE AND SUPPLY SYSTEM WHEREIN THE CUVETTE INLET ANDOUTLET ARE LOCATED ON THE BOTTOM OF THE CUVETTE Erik W. Anthon,Kensington, Calif., assignor, by mesne assignments, to Warner-LambertPharmaceutical Company, Morris Plains, NJ.

Filed Dec. 9, 1963, Ser. No. 329,201 4 Claims. (Cl. 8814) ABSTRACT OFTHE DISCLOSURE A apparatus for transferring samples to aspectrophotometer automatically and in serial fashion including aspecial cuvette constructed for automatic filling and emptying andcapable of self-cleaning from a small quantity of sample, said cuvettebeing constructed as a cylindrical housing having a bottom and a plungeradapted to reciprocate in said housing so that the plunger and bottomwall fit in matching configuration and the cuvette operates between thesubstantially zero volume and the filled volume, a filling duct directedvertically through the bottom wall so as to direct incoming sampletoward the plunger and a separate and distinct outlet duct extendingthrough the bottom of the chamber for removing fluid therefrom, saidfilling and exit ducts preferably having their ends terminating at theinner surface of the bottom wall of the chamber and in concentricrelation.

The present invention relates to improvements in an analytical cuvetteand method of filling same and more particularly to a cuvette adapted tofit in operative position in a sensing device for an automatedanalytical systern and an automatic supply system for moving sample intoand out of the cuvette.

In certain types of analytical procedures, large numbers of routinedeterminations must be carried out. Particularly applicable are thoseemployed in biochemical, physiological and medical laboratories wherematerials such as blood serum, tissue serum, urine, or other materialsare tested and/or analyzed. Such tests are particularly suited forautomation because the analytical procedures are repeated a large numberof times.

In accordance with many of these procedures, the final measurement ismade by a suitable sensing device capable of measuring a physicalproperty which is indicative of the analytical results to be found. Forexample, many procedures call for the addition of reagents which developcolor in an amount depending upon the concentration of a constituent inthe sample. When the apparatus is to be fully automated and to beprogrammed to carry out such a procedure, it is necesary to providemeans for holding the sample in operative position in the sensing deviceand to provide an automatic means for supplying samples to be determinedin an orderly and known fashion to said means.

A cuvette and an automatic supply system therefor capable of achievingthese results is the subject of the copending United States patentapplication, Ser. No. 228, 337 entitled, Cuvette and Supply SystemTherefor, filed Oct. 4, 1962, now patent No. 3,225,645 by Hans Baruchand Erik W. Anthon, the inventor in the present application, andassigned to the same assignee as the present invention. While this priorapplication describes and claims an excellent cuvette system, it hasbeen found that improvements can be made on the system, and the presentinvention is directed to such improvements.

In the device containing the analytical cuvette of the invention, thesensing device may be any type of instrument adapted to measure thephysical properties of a substance contained in operative positiontherein. For example, physical properties such a color, ultravioletabsorption, fluorescence, or any other property utilized for analyticalpurposes may be measured with a sensing device adapted to measure suchproperties. The invention is particularly applicable for use with aspectrophotometer or colorimeter which measures color developments orsimilar optical properties utilized in analytical procedures.

The supply system may be any system capable of moving sample, which hasbeen treated according to the necessary procedure for measurement of thephysical property, from a site or location in the apparatus to a cuvettein the sensing device. However, it is important that the supply systembe fully automated with its automation properly synchronized into thesystem, and it is also important to provide a system in whichcontamination by prior samples handled is completely eliminated.

In order to eliminate or sufiiciently reduce this contamination fromprior samples, it is necessary to provide a system in which the cuvetteand supply ducts are cleangd. The present invention is directed toimprovements in the cuvette construction and supply ducts which gives abetter cleaning situation and allows for a simpler supply system thanthat shown in detail in the prior application cited above.

Thus it is the primary object of this invention to provide an improvedanalytical cuvette and method of filling same of the character describedwhich is capable of moving treated sample into and out of position in asensing system where a reading which gives the analytical result ismade.

Another object of the invention is the provision of an improved methodand apparatus for filling a cuvette with a series of samples in whichthe cuvette is Washed clean of prior sample by a portion of sample beingbrought in for measurement, and then the remainder of the sample to bemeasured is brought into position for accurate determination.

A further object of the invention is to provide an improved cuvettewhich is capable of continued automatic operation over a long period oftime and which is resistant to corrosive chemicals and atmospheresusually found in a laboratory.

A still further object of the invention is to provide an improvedanalytical cuvette and supply system of the character described which iseasily constructed, simple in operation and reliable and accurate in theresults obtained.

Further objects and advantages of the invention will be apparent as thespecification progresses, and the new and useful features of myanalytical cuvette and method of filling the same will be fully definedin the claims attached hereto.

In its broad aspect, the invention provides a cuvette which has a pumpassociated therewith for moving treated sample into the cuvette chamberand moving treated sample out of the cuvette chamber in a simplifiedmanner while solving the problem of undue cross-contamination. In theprocess form, the invention operates to utilize the first receivedsample material to rinse away prior contaminants and then use the lastreceived sample in the container to obtain a reading which issubstantially unaffected by prior samples handled.

In its preferred form, the cuvette is constructed with a pump as anintegral part thereof, with a cylinder and plunger operating within thecylinder with the cylinder serving as the container for the sample to bemeasured. In this way, the pump is used to bring in and remove samplefrom the cuvette. In order to provide the proper flow of liquid, twolines communicate into the cuvette, one of which brings in the samplewhile the other takes sample out toward a drain. In order to provideproper flow of liquid, a suitable valving system is used which acts incooperation with the pump to insure the necessary flow of liquid.

In accordance with the present invention it is possible to carry out themethod of the invention with a minimum of apparatus, and it is preferredto utilize a pair of pump units which are driven by air pressure and avalve system which operates all pumps and regulates the associated fluidlines in one operation. In this way, the valve operations are automatedby any suitable control systems and the operation of the system followsfrom the positioning of the valves.

Specifically, the cuvette is designed to have the entrance conduit andthe exit conduit for the sample terminate in substantially juxtaposedlocations in the cuvette chamber so that efficient washing is effectedas sample moves through the cuvette chamber. In its preferred form, theinlet conduit is concentrically disposed within the exit conduit, andthe washing or rinsing is accomplished when the cuvette chamber is atminimal volume, the volume of the chamber being varied by a plunger orthe like.

The preferred form of the invention is illustrated in the accompanyingdrawings forming part of this description, in which:

FIGURE 1 is a schematic showing of a complete dual system for loading acuvette with sample to be determined, and loading another cuvette with ablank sample so that a difference comparison can be made in aspectrophotometer; and

FIGURE 2, an enlarged cross-sectional view of a portion of the apparatusillustrated in FIGURE 1 showing certain of the components in greaterdetail.

While only the preferred form of the invention is shown in the drawings,it should be understood that various changes or modifications may bemade within the scope of the claims attached hereto Without departingfrom the spirit of the invention.

Referring to the drawing in greater detail, there is shown in FIGURE 1 aliquid handling system 11 which comprises a cuvette 12, a sample site 13and a pump system 14 for moving sample from the sample site into thecuvette and for removing sample from the cuvette. In addition, thesystem shown also comprises a cuvette 16 adapted to receive blank samplefrom a blank sample container 17, and a pump system 18 for moving blanksample into and out of the cuvette 16 Thus, a dual system is shown withthe cuvette 16 being similar to the cuvette 12, and the pump system 18being similar to the pump system 14. Accordingly, the description givenbelow for the cuvette 12 and the pump system 14 is also applicable tocuvette 16 and pump system 18, and the similar parts of the two systemsare similarly numbered.

In the system shown, the cuvette 12 and the cuvette 16 are both carriedin a spectrophotometer 19 which contains a pair of photosensing tubes 21and means (not shown) for providing light rays as indicated by arrows 22through the sample and blank simultaneously and to the photosensingtubes 21.

As here shown, the cuvettes 12 and 16 each comprise a cylindricalhousing 23 made of precision bore glass tubing, a bottom plug 24 whichhas a conduit assembly 26 therein, and a plunger 27. The bottom plug ispreferably made of Teflon and has a central opening or duct 28 whichserves as the outlet conduit for cuvette chamber 29. Inlet tube 31 ispreferably concentrically disposed within duct 28 and has its outlet endsubstantially even with the upper surface of the bottom plug 24 as bestseen in FIG- URE 2.

The conduit assembly 26 comprises ring shaped fittings 32 and 33 andclosure fitting 34 to define a chamber 25 which serves as a part of theexit duct. The exit duct then continues through tube 36 in the fitting32 as shown. Inlet tube 31 passes through fitting 33 and is carriedtherein in 4 tight gripping relation where it is joined with supply line30. Tube 31 is preferably stiff enough to retain its shape and positionduring operation. As here shown, tube 31 is also preferably cut at anangle at its end.

The fluid line 30 has a stiff probe section 40 at its end and the lineis smooth without joints or irregularities that may cause holdup ofliquid and cross-contamination between sarnples. The use of valves inline 30 is also avoided for the same reason. V

The plunger 27 is constructed to slide sealingly'within the housing 23,and has an end shaped to match the configuration of the bottom plug 24.In this way, the plunger acts as a pump that draws liquid into andexpels liquid out of the chamber 29. The fit is also tight enough thatthe plunger keeps the walls of the glass tube clean and removes any filmthat might otherwise collect thereon. Plunger 27 is also-preferablyconstructed with grooves 38 which help provide an air-tight seal betweenthe Teflon plunger and glass housing.

The plunger is moved up and down by an air cylinder 39 through a rockerarm 41 which is connected to a piston arm 42 of a piston 43 carried inthe air cylinder. A plunger 44 is also attached to the piston 43, andthis plunger fits into a chamber 46 defined by a housing 47. Thus theplunger 44 and chamber 46 serves as a pump means 48. It is seen fromFIGURE 1 of the drawing that air cylinder 39 simultaneously operates thepump means 48 and plunger 27. With this operation, the pump means takesin liquid while the cuvette chamber is emptied of its contents, andwhile the cuvette takes in liquid the pump means expels liquid. Thisoperation is important because pump means prevents the cuvette fromemptying its contents back through the probe 40. In order to achievethis result, the pump means should be constructed to draw in a largeramount of liquid than is discharged by the cuvette.

In order to achieve the improved method of loading the cuvette chamberaccording to the invention it is important that the capacity of the pumpmeans exceeds the added volume of the cuvette chamber and supply line30. The. excess amount corresponds to the volume of sample used to washthe cuvette, and therefore, this excess should be a positive amount ofsufiicient for the purpose. In a typical example the capacity of thepump means is 2.5 ml., the cuvette chamber 0.5 ml. and the capacity ofthe supply line 1.5 ml. This provides an excess of sample for washing of0.5 ml., and such amount is sufficient to achieve sufiicient rinsingbecause the cuvette chamber is reduced to nearly a zero volume duringthe rinse operation. In this example, the carryover between samples isof the order of 0.2%, and this amount is generally within the requiredtolerance.

Air pressure or vacuum is supplied to the air cylinders 39 through aline 49 which is controlled by a valve 52. Flow of fluids in theremainder of the system is also effected by the valve 52 as will be morefully described below.

In general, valve 52 may be any valve capable of providing the desiredchanges in the fluid lines connected thereto, and the size of the valvewill vary somewhat on the system utilized. For example, a dual system isshown in FIGURE 1. However, it will be appreciated that a single systemcould be used, and in such a case certain of the valve positions may beeliminated. As here shown, valve 52 comprises a cylindrical valve unit53 having passages therein adapted to align with passages in a valvemember 54 sliding within the cylindrical valve unit 53.

Valve member 54 is positioned to provide the communication of liquidlines and air lines as shown, or to provide the communication shown inphantom. Adjustment of the position is obtained by reciprocating motionof valve member 54 which in turn is moved by a frame 56. Frame 56 ismoved by an eccentric 57 driven by a motor (not shown) at halfrevolutions. Control for the operation of the valve positioning of theprobe in the samples and readout by the spectrophotometer is preferablyobtained by an electric control system such as a simplified form of thatshown in the copending application cited above on which the presentinvention is an improvement.

As indicated above, any valve capable of achieving the desired resultmay be used, provided it gives the desired number of fluid linepositions, and may be programmed automatically to carry out the pumpingsteps" required. These .pumping steps include transfer of sample intothe cuvette, removal of the sample from the cuvette, and cleaning of thecuvette. A preferred valve and mechanism for" operating the same isdescribed and claimed in the copending United States patent applicationSer. No. 183,506, filed Mar. 29, 1962, now Patent No. 3,119,538,entitled 'Valve by Erik W. Anthon, the inventor in the presentapplication.

The probes 40 are preferably constructed of a material having ahydrophobic surface and therefore are substandaily free of samplematerial after the passage of sample material therethrough. However, ifdesired, the probes 40 may be moved to a wash site and then to the nextsample for pumping sample material into the cuvette.

Any suitable transfer device may be used to effect movement of theprobes such as a device moving frame 58, which carries the probes in areciprocating motion as indicated by arrow 59, while the containers 61are moved by independent conveying means (not shown). Examples oftypical devices capable of effecting the desired transfer of the probemay be found in the copending patent application of Erik W. Anthon, theinventor in the present application, Ser. No. 61,260, entitled,Materials Handling Apparatus, filed Oct. 7, 1960, now Patent No.3,178,266.

In operation, the cycle ends with the cuvette plunger being up and thepump plunger being down after readout of the previous sample. The valveis positioned as shown with the pump cylinder being connected to thedrain through lines 62, valve 52 and lines 63 while the air cylinder 39is connected to the pressure side of air compressor 64 through lines 49,valve 52 and pressure line 66.

The next sample and reference blank are positioned at the sample siteand the probes 40 lowered into position. The valve is then switched tothe opposite position as shown in phantom to connect air cylinder 39 tothe suction line 67 of the compressor. At the same time, lines 62 of thepump means 48 are connected to exit lines 36 of the cuvettes. With thisvalve change the air cylinders cause the pump plungers to go up and thecuvette plungers to go down. 1

Using the volumetric values of a typical example, each pump means drawsin a volume of 2.5 ml. while the cuvette plunger goes down and drivesout a volume of 0.5 ml. Thus the differential volume of 2.0 ml. is drawnin through the probe and line 30. Since the probe and supply line onlyholds 1.5 ml., 0.5 ml. of fresh sample passes through the supply lineand the cuvette into outlet duct 28.

In the preferred construction shown, the sample is directed into thecuvette in a stream which impinges on the bottom of plunger 27 to givean excellent rinsing action. In this way a small amount of sample isexpended in rinsing and an economy of sample is obtained as well as asimplified operation. For example a sample as small as 1.0 m]. could beused since only 0.5 ml. is used for rinsing and 0.5 ml. is used forreadout. In this case and any other case where less than 2.0 ml. ofsample is available, air is drawn into the probe, but this air remainsat the probe end because of the small diameter supply line used.

The probe is then removed from the sample container and the valve isswitched back to the position shown in FIGURE 1 which closes tube 36leading from the cuvette, provides a connection between pump chamber 46and the drain, and provides pressure in air cylinder 39. This causes thepump plunger to go down and expel the pump contents to drain whilelifting the cuvette plunger and drawing in sample from supply line 30.After this operation, the cuvette chamber is filled and a read-out ismade.

After read-out, the cycle is repeated to obtain a determination for thenext sample. In a typical unit, cycle time is only 5 seconds plus thetime required for the read-out on the spectrophotometer. This rapidoperation is achieved because only two strokes are needed for eachcycle. It may be observed that in the procedure described, at least 0.5ml. of air is drawn into the probe. This air slug is valuable in keepingsample slugs separated in the supply line so as to avoidcross-contamination by diffusion between adjacent samples.

As best seen in FIGURE 2, the cuvette is adapted to be held in operativeposition in the housing 68 of spectrophotometer 19 or the like, andvarious sizes of cuvettes may be used if desired. The spectrophotometercomponents are carried in the same housing as the pumping system of thisinvention, and the holding means for the cuvette comprises an opening 69containing a V-shaped wall 70 against which the cuvette is clamped. Ingeneral, this clamping may be effected by any suitable means such asthat shown in the prior application of Hans Baruch and the presentinventor cited above.

The rocker arm 41 which operates between piston arm 42 of the pump means48 and plunger 27 of the cuvette may be easily removed from the plunger27 so as to effect a change of cuvettes. This arm is also carried in theend of the cuvette plunger by the cross slot 71 as best seen in FIGURE 2so that leverage may be effected with various sizes of cuvettes. Inaddition, rocker arm 41 may be positioned at any one of a number ofpivots.

As here shown, rocker arm 41 contains four holes 72, 73, 74 and 75 whichserve as pivots for fulcrum means 76. Fulcrum means 76 is in the form ofa pin which fits through one of the holes 72 through 75 and into acorresponding hole in support block 77. Thus the position shownillustrates a short stroke with a small cuvette. In order to obtain alonger stroke with the same cuvette, pivot pin 76 is moved over to fitthrough hole 73 of rocker arm 41 and into hole 78 in support block 77.In order to use a larger cuvette, a larger cuvette is inserted intoposition and the rocker arm 41 is rotated so that hole 75 of the rockerarm is aligned with hole 79 in the support block. In this way, the pivotmay be at 73 with the pin in hole 78, at 74 with the pin located asshown or at 75, or with the pin fitting at 79. Thus the length of thestrokes of the plunger 27 may be varied as desired with different sizesof cuvettes and different amounts of sample available.

In the description given above, a filling cycle is described asconsisting of two pump strokes. This gives satisfactory results and isthe fastest cycle. However, it has often ben found more favorable tooperate the system with four pump strokes using a lower ratio betweenthe displaced volumes of the pump and cuvette. The operating data fortypical four stroke operations are given in the tables below. As usedtherein, the terms downstroke and upstroke refer to the cuvette plungermovement The operation was tested and it was found that 2.0 ml. ofsample were sufficient. Although 3.1 ml. are pumped to drain 0.4 ml.represents air taken in during the last upstroke when sample is movedfrom the probe to the euvette, and it is possible to allow up to about0.7 ml. of air to enter the probe during the second downstroke becausethe probe tube holds more liquid than required to fill the cuvette.

Table I above illustrates a procedure for low carryover and Table IIbelow illustrates a procedure where less sample is available. In TableII, the system is designed for 1.9 ml, of sample, but actual testsindicate 1.2 ml. is suflicient. In order to change the ratio, of cuvettevolume to pump volume, the pivot pin 76 is moved to holes 73 and 78, andthe threaded head 81 on plunger 42 is adjusted. The limits of thestrokes are determined :by the bottom positions of the pump plunger andcuvette plunger. With the ratio fixed by the rocker arm, the totalvolumes are adjusted through this nut. After the change, the pump volumeis 1.15 ml., and the operative values are given in Table II.

TABLE II Pump Cuvctte Probe Diarnter (LD. mm 1l.1 6.35 1.25. Length orstroke, mm 11.9 12.7 1,000. Volume,ml 1.15 0.4 1.2. 1. Downstroke, ml1.15 in. 0.4 out 0.75 in. 2. Upstroke, ml 1.15 drain 0.4 in 0.4ir 1. 3.Downstroke, ml 1.15 in- 0.4 out 0.75 in. 4. Upstroke, ml 1.15 drain 0.4in- 0.4 air.

When sufiicient sample is available a longer light path through thesample is obtained by using a larger cuvette and reversing the rockerarm as explained above. A typical operation using a larger cuvette isgiven in Table III below. In this run, 2.7 ml. of sample represents themini- The apparatus may be constructed of any suitable materials whichare chemically resistant, but I prefer to use precision bore glass tubesfor the cuvettes, and to use a Teflon plunger and plug therein. Othermaterials which are preferably used include polypropylene tubing andViton rubber O-rings on the pump pistons. These materials are eminentlysatisfactory in providing long wear and resistance to chemical attack.

From the foregoing description, it is seen that I have provided animproved self-cleaning, fully automated system for moving samples intoreading position in a onvette, and for removing said samples from thecuvette and cleaning same for the next operation. It is also seen that Ihave provided an improved form of cuvette, which is simple in operationand capable of rapid accurate operation with a minimum of sample.

I claim:

1. An apparatus for transferring individual samples to a sensing devicefrom a sample site where samples are provided in vone-at-a-timefashion," comprising a cuvette in the sensing device for receiving asample,-'said cuvettehaving radiation permeable walls and a plunger fordraw ing in and expelling, fluid from the cuvette, a pump for collectingliquids removed from the cuvette, a first ductv in communication withthe cuvette positionable-tore: ceive sample from the sample site, asecond duct in com: munication with the cuvette chamber and the pump,said first duct and said second duct each having a separate and distinctend thereof terminating at the inner surface of the bottom wall of thecuvette chamber, and ,valve means for controlling the operation of thecuvette plunger, pump and the opening in the second duct, said firstduct being oriented to direct its fluid towardthe plunger, and said pumpbeing formed to remove .fiuid from the cuvette at a rate greater thanthe displacement of the plunger in the cuvette whereby a new sample isdirected toward the plunger as a prior sample is removed to Wash awayold sample and makethe cuvette more re ceptive to receipt of the newsample. p I

2. The apparatus for transferring samples to a sensing device defined inclaim 1, in which the end portion of the first duct terminating in thecuvette chamber is located inside the passage at the end portion of thesecond duct.

3. The cuvette defined in claim 1, in which the ends of the first andsecond duct are in'concentric alignment.

4. A cuvette adapted to receive and hold a liquid sample in automatedanalytical equipment in position for further operation thereon,comprising a housing having side walls and a bottom defining a chamberfor holding liquids, said walls being formed for accommodating saidfurther operation, a plunger adapted to reciprocate in said housing,said plunger having side walls and a bottom Wall in matchingconfiguration with said housing, a first duct having a passage incommunication with said chamber through said bottom and extendingsubstantially to the inner surface of said bottom, and a second duct incommunication with said chamber through said bottom, said second ducthaving a passage separate and distinct from the passage of said firstduct and extending substantially to the inner surface of said bottom,and said first duct having its end thereof entirely inside of thepassage of the second duct at the bottom of the cuvette, with thepassage of the first duct being directed toward the plunger wherebysample moving through the first duct is adapted to be directed againstthe plunger during at least a part of the operation thereof to wash theplunger.

References Cited UNITED STATES PATENTS 12/1965 Baruch et al.

3/1959 Bergson.

